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HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [LSC and LSK]

ABSTRACT: Understanding RBPs’ molecular functions as well as their cell-type specific activity requires identification of RBPs’ direct mRNA targets. However, efforts to identify RNA targets of RNA binding proteins in stem cells have been hindered by limited cell numbers. Here we adapted the HyperTRIBE method using an RBP fused to a Drosophila RNA editing enzyme (ADAR) to allow for global mapping of mRNA targets of the RBP MSI2 in rare mammalian adult stem cells. We applied this method to identify MSI2 RNA binding targets in mouse HSPCs (Lin- Sca1+ Kit+ or LSK cells) and mouse leukemic stem cells (LSCs). MSI2 fusion with the catalytic domain of the Hyperactive ADAR mutant (MSI2-ADA) or with the dead catalytic mutant (MSI2-DCD) were overexpressed for 48 hours in LSKs and LSCs, followed by RNA-seq. As ADAR converts A to I (G), the fusion leaves a "finger-print" where MSI2 binds. MSI2-DCD and empty vector controls were used to normalize the background editing. We show that despite comparable expression of the MSI2-ADA fusion protein and endogenous MSI2 in LSCs vs LSKs, MSI2 RNA binding activity (number of targets and editing frequency) in LSCs is significantly higher than that in LSKs. This elevated RNA binding activity is independent of abundancy of the target transcripts.

ORGANISM(S): Mus musculus

PROVIDER: GSE132948 | GEO | 2020/03/11

REPOSITORIES: GEO

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HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [MOLM-13]

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HyperTRIBE uncovers MSI2 increased RNA binding activity and differential regulation in leukemic stem cells [LSK cells]

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The mRNA stability factor Khd4 defines a specific RNA regulon for membrane trafficking in the pathogen Ustilago maydis [RNA-Seq]

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Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR) [expression]

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